*Target (protein/gene name):serine/threonine protein phosphatase 2b (catalytic subunit) *NCBI Gene # or RefSeq#:156082205 *Protein ID (NP or XP #) or Wolbachia#: XP_001608591
(use Summer13D Plate for Oligos instead of ordering new ones - Dr. B 090313))
091213 - also use the GDocs/Misc/Primers/DNAWorks_OutputSummer13 to get your correct DNA Works file for Tail Primer Design
To do:
-Make homology model (Gdocs-Protocols-VirtualScreeningProtocols-HomologyModels-InstructionsProMolProject_HomologyModelversion6 (put in own folder)
-Find 10-15 positive controls & 5 negative controls
-Positive controls: binding database, ligands that stick to other similar organisms (PubChem)
-ligand prep
*Organism (including strain):Plasmodium vivax Etiologic Risk Group (see link below):Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents
http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334 *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Plasmodium vivax is geographically the most widely distributed cause of malaria in people, with up to 2.5 billion people at risk and an estimated 80 million to 300 million clinical cases every year--including severe disease and death. Despite this large burden of disease, P vivax is overlooked and left in the shadow of the enormous problem caused by Plasmodium falciparum in sub-Saharan Africa. The technological advances enabling the sequencing of the P vivax genome and a recent call for worldwide malaria eradication have together placed new emphasis on the importance of addressing P vivax as a major public health problem. However, because of this parasite's biology, it is especially difficult to interrupt the transmission of P vivax, and experts agree that the available methods for preventing and treating infections with P vivax are inadequate. It is thus imperative that the development of new methods and strategies become a priority. Advancing the development of such methods needs renewed emphasis on understanding the biology, pathogenesis, and epidemiology of P vivax. This Review critically examines what is known about P vivax, focusing on identifying the crucial gaps that create obstacles to the elimination of this parasite in human populations.
Essentiality of this protein: This protein is essential on the 6th day of development, where a knocked-out gene will cause P. vivax to loose fitness in bloodstream and procyclic forms. Complex of proteins?: No Druggable Target: Yes, Druggability index (0-1) of 0.8 *EC#: 3.1.3.16
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2IE4_C
-- For Homology Model option:
---- Query Coverage: 72%
---- Max % Identities: 55%
---- % Positives 73%
---- Chain used for homology: Chain C
Current Inhibitors: Calcineurin was the only inhibitor found for serine/threonine protein phosphatase 2b (catalytic subunit), however, it was only studied in Humans, not Plasmodium vivax. Expression Information (has it been expressed in bacterial cells): Yes Purification Method: microcystin-Sepharose affinity chromatography
Image of protein (PyMol with features delineated and shown separately):
Figure 1 shows Chain C, Structure Of The Protein Phosphatase 2a Core Enzyme Bound To Okadaic Acid , which has the highest amino acid sequence overlap as serine/threonine protein phosphatase 2b.
*Amino Acid Sequence: MEPLPNPKNDRQVKDVEPPPAKPLSLELLYPNGTDEPPDYKALRDHLKKEGRIRKEDCLDIIKRVIDIVS NEPNLLRLQDPITIVGDIHGQYYDFLKLLEVGGNPDNTQFLFLGDYVDRGSFSIEVLLLLYALKINFPHK IWLIRGNHECRQMTSFFNFRDECEYKYDMVVYYAFMESFDTIPLSAVINGKFLAVHGGLSPQLVLLNQIC SFTRFQEPPRSGIFCDILWADPIDEDKEEHTIQTESYFPNDIRGCSYFFGYNAATTFLEKNGLLSIIRAH EAQLEGYKMHQTNLKTGFPIVITIFSAPNYCDVYNNKGAVLKFDSNTLNIQQFSFSPHPYHLPNFMNLFT WSLPFVSEKVTEMLYCILNSSVNQSDEGVKDVVLPAEVLQIISYIEENNVKLEGMTLSGGGGATAGAAST GATEGSPSSQRKEALFKEGCFHSGASKEGGALGTTSPAAATATTQQMAAQGEQPAHLHTDDAQASKERSD ALRKKVQSVGRLMRVFRTLRKENELIVQLKGCSPGYRIPVGLLLQGKEGLENELEKFTKAKQIDSINEKR PPNE *length of your protein in Amino Acids: 564 Molecular Weight: 63 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 46925 M-1 cm-1 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*NCBI Gene # or RefSeq#:156082205
*Protein ID (NP or XP #) or Wolbachia#: XP_001608591
(use Summer13D Plate for Oligos instead of ordering new ones - Dr. B 090313))
091213 - also use the GDocs/Misc/Primers/DNAWorks_OutputSummer13 to get your correct DNA Works file for Tail Primer Design
To do:
-Make homology model (Gdocs-Protocols-VirtualScreeningProtocols-HomologyModels-InstructionsProMolProject_HomologyModelversion6 (put in own folder)
-Find 10-15 positive controls & 5 negative controls
-Positive controls: binding database, ligands that stick to other similar organisms (PubChem)
-ligand prep
*Organism (including strain): Plasmodium vivax
Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents
http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Plasmodium vivax is geographically the most widely distributed cause of malaria in people, with up to 2.5 billion people at risk and an estimated 80 million to 300 million clinical cases every year--including severe disease and death. Despite this large burden of disease, P vivax is overlooked and left in the shadow of the enormous problem caused by Plasmodium falciparum in sub-Saharan Africa. The technological advances enabling the sequencing of the P vivax genome and a recent call for worldwide malaria eradication have together placed new emphasis on the importance of addressing P vivax as a major public health problem. However, because of this parasite's biology, it is especially difficult to interrupt the transmission of P vivax, and experts agree that the available methods for preventing and treating infections with P vivax are inadequate. It is thus imperative that the development of new methods and strategies become a priority. Advancing the development of such methods needs renewed emphasis on understanding the biology, pathogenesis, and epidemiology of P vivax. This Review critically examines what is known about P vivax, focusing on identifying the crucial gaps that create obstacles to the elimination of this parasite in human populations.
Essentiality of this protein: This protein is essential on the 6th day of development, where a knocked-out gene will cause P. vivax to loose fitness in bloodstream and procyclic forms.
Complex of proteins?: No
Druggable Target: Yes, Druggability index (0-1) of 0.8
*EC#: 3.1.3.16
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Spectrophotometric.
Reagents: 200 mM Tris HCl Buffer, pH 7.5 at 30°C (Prepare 100 ml in deionized water, using Trizma Base, Sigma Prod. No. T-1503. Adjust to pH 7.5 at 30°C with 1 M HCl.)
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/calcineurin.pdf
-- link (or citation) to paper that contains assay information
http://www.ncbi.nlm.nih.gov/pubmed/23233447
-- List cost and quantity of substrate reagents and supplier
http://www.sigmaaldrich.com/catalog/product/sigma/c5207?lang=en®ion=US
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2IE4_C
-- For Homology Model option:
---- Query Coverage: 72%
---- Max % Identities: 55%
---- % Positives 73%
---- Chain used for homology: Chain C
Current Inhibitors: Calcineurin was the only inhibitor found for serine/threonine protein phosphatase 2b (catalytic subunit), however, it was only studied in Humans, not Plasmodium vivax.
Expression Information (has it been expressed in bacterial cells): Yes
Purification Method: microcystin-Sepharose affinity chromatography
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence:
MEPLPNPKNDRQVKDVEPPPAKPLSLELLYPNGTDEPPDYKALRDHLKKEGRIRKEDCLDIIKRVIDIVS NEPNLLRLQDPITIVGDIHGQYYDFLKLLEVGGNPDNTQFLFLGDYVDRGSFSIEVLLLLYALKINFPHK IWLIRGNHECRQMTSFFNFRDECEYKYDMVVYYAFMESFDTIPLSAVINGKFLAVHGGLSPQLVLLNQIC SFTRFQEPPRSGIFCDILWADPIDEDKEEHTIQTESYFPNDIRGCSYFFGYNAATTFLEKNGLLSIIRAH EAQLEGYKMHQTNLKTGFPIVITIFSAPNYCDVYNNKGAVLKFDSNTLNIQQFSFSPHPYHLPNFMNLFT WSLPFVSEKVTEMLYCILNSSVNQSDEGVKDVVLPAEVLQIISYIEENNVKLEGMTLSGGGGATAGAAST GATEGSPSSQRKEALFKEGCFHSGASKEGGALGTTSPAAATATTQQMAAQGEQPAHLHTDDAQASKERSD ALRKKVQSVGRLMRVFRTLRKENELIVQLKGCSPGYRIPVGLLLQGKEGLENELEKFTKAKQIDSINEKR PPNE
*length of your protein in Amino Acids: 564
Molecular Weight: 63 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 46925 M-1 cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*CDS Gene Sequence:
ATGGAGCCTCTACCAAACCCTAAGAACGATAGACAAGTGAAAGACGTGGAACCCCCACCAGCTAAGCCGC TGAGCCTGGAGCTGCTGTACCCGAACGGCACGGACGAGCCGCCAGACTACAAGGCGCTGCGCGACCACCT GAAGAAGGAGGGGCGCATCCGCAAGGAGGACTGCCTAGACATCATCAAGAGGGTGATCGACATCGTGAGC AACGAGCCAAACCTGCTGCGGCTCCAAGACCCAATAACGATCGTGGGGGACATCCACGGCCAGTACTACG ATTTTCTGAAGCTACTGGAAGTGGGAGGAAACCCAGATAACACTCAGTTCCTTTTCCTGGGCGACTACGT AGACAGAGGTTCCTTCAGTATAGAGGTCCTGCTCCTCCTCTACGCCCTCAAAATTAATTTCCCACATAAA ATATGGCTCATAAGAGGAAATCACGAGTGTAGGCAGATGACCTCCTTCTTCAATTTTAGAGACGAATGCG AATACAAATACGATATGGTTGTGTATTATGCCTTTATGGAATCCTTTGATACGATCCCCTTATCGGCTGT AATCAATGGGAAGTTCCTAGCCGTCCATGGGGGGTTGTCTCCTCAACTGGTACTCCTCAATCAGATATGT TCCTTCACGAGATTCCAAGAACCCCCACGGTCAGGGATTTTCTGTGACATTTTATGGGCGGACCCAATTG ACGAAGATAAAGAGGAGCATACCATTCAGACGGAATCCTACTTCCCCAATGACATCCGAGGGTGTAGCTA CTTCTTTGGCTATAATGCTGCCACCACCTTTTTAGAGAAGAACGGGTTACTCTCCATTATAAGAGCTCAT GAGGCACAGTTGGAGGGATACAAAATGCATCAAACCAATTTGAAGACCGGCTTCCCCATAGTCATCACCA TATTTTCTGCCCCTAATTATTGTGACGTTTATAATAACAAGGGGGCTGTACTCAAATTCGATAGCAACAC GTTGAACATCCAACAGTTTAGCTTTTCCCCCCACCCGTATCACCTCCCCAATTTTATGAACCTCTTCACC TGGTCTCTCCCCTTTGTTAGCGAGAAAGTCACAGAAATGCTCTACTGCATTTTAAACTCCAGCGTCAACC AATCGGATGAGGGGGTGAAGGACGTCGTGCTTCCTGCGGAGGTCCTCCAGATTATTAGCTACATAGAGGA GAACAATGTCAAGCTGGAGGGGATGACTCTAAGTGGTGGTGGTGGTGCTACTGCCGGTGCTGCTTCTACC GGTGCTACCGAGGGGTCCCCCTCCTCACAGAGGAAGGAAGCCCTTTTCAAGGAGGGCTGCTTCCACAGTG GGGCATCCAAAGAAGGGGGGGCATTGGGAACAACCTCCCCCGCTGCCGCTACTGCGACTACGCAGCAAAT GGCCGCCCAGGGGGAGCAACCGGCCCACCTGCACACCGATGACGCGCAGGCGTCCAAGGAACGGTCCGAC GCCCTCAGGAAAAAGGTTCAGTCAGTTGGTCGCCTGATGAGAGTGTTTAGGACGCTGCGCAAGGAGAACG AGTTGATTGTGCAGCTCAAGGGGTGCAGCCCCGGCTACCGCATCCCCGTGGGCCTCCTCCTGCAGGGCAA GGAGGGGCTGGAGAACGAGCTGGAGAAGTTCACCAAGGCCAAGCAGATCGACAGCATAAATGAGAAGCGG CCGCCGAACGAGTAG
*GC% Content for gene: 52.3%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
https://docs.google.com/file/d/0B4O2KqKh2q_-a1R3R1psN0ZWS0U/edit?usp=sharing
Primer design results for 'tail' primers (this is just 2 sequences):
References:
http://www.ncbi.nlm.nih.gov/pubmed/19695492
http://www.ch.embnet.org/cgi-bin/TMPRED_form_parser
http://www.tdrtargets.org/targets/view?gene_id=265333
http://www.ncbi.nlm.nih.gov/protein/156082205?report=fasta
http://www.ncbi.nlm.nih.gov/nuccore/156082204?report=fasta
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/calcineurin.pdf
DVGGDIMNNDYIFLGDYVDRGYNSVETFEYLLLLKLLFPKNITLLRGNHESRQITTVYGF 127